Recent evidence indicates that the reversible carboxymethylation of proteins plays an important role in the regulation of numerous cellular functions. The overall goal of the proposed research is to provide an increased understanding of the role of protein carboxymethylation in neuronal function. Initial experiments will focus on the mechanism of regulation of protein carboxymethylase from rabbit brain. Current evidence suggests that this enzyme may be subject to regulation by second messengers. Therefore, the effects of cyclic-AMP, cyclic-GMP, and Ca (plus) on protein carboxymethylase activity in brain cytosol will be studied. If these agents appear not to be activators, a search for other possible endogenous activators in brain cytosol will be made. In order to identify specific methyl accepting substrates for protein carboxymethylase, cytosol and lysed synaptosomes from identified brain regions will be subjected to in vitro methylation with (3H-methyl)-S-adenosylmethionine and then separated by polyacrylamide gel electrophoresis. Substrates which are found to be unique to brain will become candidates for purification and further characterization. Brain slices will be exposed to various neurotransmitters and depolarizing agents to determine if there is any effect on the state of methylation of previously characterized substrates. Antibodies to purified protein carboxymethylase and its substrates will be made and used to study the regional, cellular and subcellular distribution of these proteins by immunocytochemical techniques and by radioimmunoassay.